The sum of the real-world conditions argues strongly that smallpox could be used as a particularly devastating weapon of bioterrorism. There is an urgent medical and national need to design a novel anti-smallpox therapy and to make it available in addition to the existing preventive measures, to the US population. The comprehensive and integrated "key national priority" program we are proposing meets directly the stated objectives set forth by RFA AI-03-017 and is focused on a rational structure-based drug design for a smallpox therapy. Our strategies towards developing a smallpox therapy are guided by the development of the only successful and selective anti-viral therapy, the AIDS protease inhibitor "cocktail". There are only two proteinases (H1L and K7L) encoded by the variola genome, both of which are involved in the processing of viral protein precursors and virion assembly. Our work will generate the first and the only set of protease inhibitors applicable as a rapid response to a smallpox outbreak/attack. The Burnham Institute scientists participating in this effort are uniquely qualified to accomplish the program successfully. Our team includes nationally and internationally known experts on metallo- and cysteine proteinases, internationally recognized structural biologists, and experts on the cutting edge of drug design. We will use several independent, albeit overlapping pipelines, to generate the inhibitors and to ensure the overall success of the program. Our aims are: (I) to express, purify and characterize the recombinant metalloproteinase H1L and the cysteine proteinase K7L of vaccinia/variola, (lI) to identify optimal peptide substrates for the recombinant proteinases and then to derivatize these substrates into lead peptidic antagonists, (III) to identify drug-like small molecule inhibitors of the variola proteases by screening combinatorial chemical libraries, (IV) to determine, at the atomic resolution level, the structure of the individual H1L and K7L proteases and their structures bound to lead antagonists and optimal peptide substrates, (V) to increase, through iterative rational structure-based drug design, the selectivity, potency, and safety of the selected drug leads, (VI) to examine the efficacy and safety of the drug candidates in vitro and in cell-based assays of vaccinia viral replication. When appropriate benchmarks are met, the drug candidates will be delivered to the government for continued testing and refinement involving the variola virus. [unreadable] [unreadable]